LOCUS       sequence-231562-        2183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA targeting position 6 in pColE1_70a_deGFP_KanR with one
            mismatch in the seed region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2183)
  AUTHORS   Collias D, Marshall R, Collins SP, Beisel CL, Noireaux V
  TITLE     An educational module to explore CRISPR technologies with a 
            cell-free transcription-translation system.
  JOURNAL   Synth Biol (Oxf). 2019 Jan 21;4(1):ysz005. doi: 
            10.1093/synbio/ysz005. eCollection 2019.
  PUBMED    32995532
REFERENCE   2  (bases 1 to 2183)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Synth Biol 
            (Oxf)."; date: "2019-01-21"; volume: "4(1):ysz005. doi"; pages: " 
            10.1093/synbio/ysz005. eCollection 2019"
COMMENT     Sequence Label: pColE1_sgRNA_M1S_AmpR
FEATURES             Location/Qualifiers
     source          1..2183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..104
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        125..200
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(462..1253,1254..1322))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1085..1104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1323..1427)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1530..2118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2019..2038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc cgagagcatc gatcctagca
       61 tggatgcatt tgacagctag ctcagtccta ggtataatgc tagcggtaaa attgtcaaca
      121 cgctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      181 aagtggcacc gagtcggtgc tttttttccg gcttatcggt cagtttcacc tgatttacgt
      241 aaaaacccgc ttcggcgggt ttttgctttt ggaggggcag aaagatgaat gactgtccac
      301 gacgctatac ccaaaagaaa aagcttgaaa gaatcgatcg atcccgaatg agtagtacag
      361 cggccgcgat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      601 cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
      961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1441 aaagtgccac ctgtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1501 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1561 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1621 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     1681 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1741 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1801 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1861 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1921 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1981 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2041 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2101 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2161 tggccttttg ctcacatgtt ctt
//
